Spatial Positioning of Spectrally Labeled Beads

ABSTRACT

Devices, systems, kits, and methods for detecting and/or identifying a plurality of spectrally labeled bodies well-suited for performing multiplexed assays. By spectrally labeling the beads with materials which generate identifiable spectra, a plurality of beads may be identified within the fluid. Reading of the beads is facilitated by restraining the beads in arrays, and/or using a focused laser.

CROSS-REFERENCES TO RELATED APPLICATIONS

The application claims the benefit of priority and is a continuation ofU.S. Non-Provisional application Ser. No. 09/827,256 filed Apr. 5, 2001,and U.S. Provisional Patent Application No. 60/195,520 entitled “Methodfor Encoding Materials with Semiconductor Nanocrystals, CompositionsMade Thereby, and Devices for Detection and Decoding Thereof,” filedApr. 6, 2000, the full disclosure of which is incorporated herein byreference.

The subject matter of the present application is related to thefollowing co-pending patent applications, the disclosures of which arealso incorporated herein by reference: U.S. patent application Ser. No.09/160,458 filed Sep. 24, 1998 and entitled, “Inventory Control”; U.S.patent application Ser. No. 09/397,432 filed Sep. 17, 1999, and alsoentitled “inventory Control”; PCT Patent Application No. WO 99/50916 aspublished on Apr. 1, 1999, entitled “Quantum Dot White and Colored LightEmitting Diodes”; and U.S. patent application Ser. No. 09/259,982 filedMar. 1, 1999, and entitled “Semiconductor Nanocrystal Probes forBiological Applications and Process for Making and Using Such Probes”.All other references cited herein are also incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention generally provides devices, compositions ofmatter, kits, systems, and methods for detecting and identifying aplurality of spectrally labeled bodies. In a particular embodiment, theinvention provides systems and methods for detecting and identifying aplurality of spectral codes generated by such bodies, especially formeasuring the results of high-throughput bead-based assay systems, andthe like. The invention will often use labeled beads which generateidentifiable spectra that include a number of discreet signals havingmeasurable characteristics, such as wavelength and/or intensity.

Tracking the locations and/or identities of a large number of items canbe challenging in many settings. Barcode technology in general, and theUniversal Product Code in particular, has provided huge benefits fortracking a variety of objects. Barcode technologies often use a lineararray of elements printed either directly on an object or on labelswhich may be affixed to the object. These barcode elements oftencomprise bars and spaces, with the bars having varying widths torepresent strings of binary ones, and the spaces between the bars havingvarying widths to represent strings of binary zeros.

Barcodes can be detected optically using devices such as scanning laserbeams or handheld wands. Similar barcode schemes can be implemented inmagnetic media. The scanning systems often electro-optically decode thelabel to determine multiple alphanumerical characters that are intendedto be descriptive of (or otherwise identify) the article or itscharacter. These barcodes are often presented in digital form as aninput to a data processing system, for example, for use in point-of-saleprocessing, inventory control, and the like.

Barcode techniques such as the Universal Product Code have gained wideacceptance, and a variety of higher density alternatives also have beenproposed. Unfortunately, these standard barcodes are often unsuitablefor labeling many “libraries” or groupings of elements. For example,small items such as jewelry or minute electrical components may lacksufficient surface area for convenient attachment of the barcode.Similarly, emerging technologies such as combinatorial chemistry,genomics research, microfluidics, potential pharmaceutical screening,micromachines, and other nanoscale technologies do not appearwell-suited for supporting known, relatively large-scale barcode labels.In many of these developing fields, it is often desirable to make use oflarge numbers of compounds within a fluid, and identifying and trackingthe movements of such fluids using existing barcodes is particularlyproblematic. While a few chemical encoding systems for chemicals andfluids have been proposed, reliable and accurate labeling of largenumbers of small and/or fluid elements remains a challenge.

Small scale and fluid labeling capabilities have recently advancedradically with the suggested application of semiconductor nanocrystals(also known as C-Dot™ particles), as detailed in U.S. patent applicationSer. No. 09/397,432, the full disclosure of which is incorporated hereinby reference. Semiconductor nanocrystals are microscopic particleshaving size-dependent optical and/or electromagnetic emissionproperties. As the band gap energy of such semiconductor nanocrystalsvary with a size, coating and/or material of the crystal, populations ofthese crystals can be produced having a variety of spectral emissioncharacteristics. Furthermore, the intensity of the emission of aparticular wavelength can be varied, thereby enabling the use of avariety of encoding schemes. A spectral label defined by a combinationof semiconductor nanocrystals having differing emission signals can beidentified from the characteristics of the spectrum emitted by the labelwhen the semiconductor nanocrystals are energized.

A particularly promising application for semiconductor nanocrystals isin multiplexed and/or high-throughput assay systems. Multiplexed assayformats would be highly desirable for improved throughput capability,and to match the demands that combinatorial chemistry is putting onestablished discovery and validation systems for pharmaceuticals. Forexample, simultaneous elucidation of complex protein patterns may allowdetection of rare events or conditions, such as cancer. In addition, theever-expanding repertoire of genomic information would benefit from veryefficient, parallel and inexpensive assay formats. Desirable multiplexedassay characteristics may include ease of use, reliability of results, ahigh-throughput format, and extremely fast and inexpensive assaydevelopment and execution.

A number of known assay formats may currently be employed forhigh-throughput testing. Each of these formats has limitations, however.By far the most dominant high-throughput technique is based on theseparation of different assays into different regions of space. The96-well plate format is the workhorse in this arena.

In 96-well plate assays, the individual wells (which are isolated fromeach other by walls) are often charged with different components, andthe assay is performed and then the assay result in each well measured.The information about which assay is being run is carried with the wellnumber or the position on the plate, and the result at the givenposition determines which assays are positive. These assays can be basedon chemiluminescence, scintillation, fluorescence, scattering, orabsorbance/colorimetric measurements, and the details of the detectionscheme depend on the reaction being assayed.

Multi-well assays have been reduced in size to enhance throughput, forexample, to accommodate 384 or 1536 wells per plate. Unfortunately, thefluid delivery and evaporation of the assay solution at this scale aresignificantly more confounding to the assays. High-throughput formatsbased on multi-well arraying often rely on complex robotics and fluiddispensing systems to function optimally. The dispensing of theappropriate solutions to the appropriate bins on the plate poses achallenge from both an efficiency and a contamination standpoint, andpains must be taken to optimize the fluidics for both properties.Furthermore, the throughput is ultimately limited by the number of wellsthat one can put adjacent on a plate, and the volume of each well.Arbitrarily small wells may have arbitrarily small volumes, resulting ina signal that scales with the volume, shrinking proportionally with thecube of the radius. Nonetheless the spatial isolation of each well, andthereby each assay, has been much more common than running multipleassays in a single well. Such single-well multiplexing techniques arenot widely used, due in large part to the difficulty in “demultiplexing”or resolving the results of the different assays in a single well.

For even higher throughput genomic and genetic analysis techniques,positional array technology has been shrunk to microscopic scales, oftenusing high-density oligonucleotide arrays. Over a 1-cm square of glass,tens to hundreds of thousands of different nucleotides can be writtenin, for example, 25-.mu.m spots, which are well resolved from eachother. On this planar test structure or “chip,” which is emblazoned withan alignment grid, a particular spot's x,y position determines whicholigonucleotide is present at that spot. Typically 3′- or 5′-labeledamplified DNA is added to the array, hybridized and is then detectedusing fluorescence-based techniques. Although this is a very powerfultechnique for assaying a large number of genetic markers simultaneously,the cost is still very high, and the flexibility of this assay islimited.

Once the masks have been written for the photolithographic process thatbuilds the particular DNA sequences into a particular location on thechip, they are fixed and the addition thereto of new markers comes at avery high price. The extremely small feature size, and the highlyparallel assay format, comes at the cost of the flexibility inherent ina common platform system such as the 96-well plates. In addition, theassay is ultimately performed at the surface of the chip, and theresults depend on the kinetics of the hybridization to the surface, aprocess that is negatively influenced by steric issues and diffusionissues. In fact, small microarray chips are not particularly suited tothe detection of rare events, as the diffusion of the solution over thechip may not be sufficiently thorough. In order to perform thehybridizations to the microarray chips more efficiently, a dedicatedfluidics workstation can be used to pump the solution over the surfaceof the chip repeatedly; such instruments add cost and time to executionof the assay.

The use of spectral barcodes holds great promise for enhancing thethroughput of assays, as described in an application entitled“Semiconductor Nanocrystal Probes for Biological Applications andProcess for Making and Using such Probes,” U.S. application Ser. No.09/259,982 filed Mar. 1, 1999, the full disclosure of which isincorporated herein by reference. Multiplexed assays may be performedusing a number of probes which include both a spectral label (often inthe form of several semiconductor nanocrystals) and one or moremoieties. The moieties may be capable of selectively bonding to one ormore detectable substances within a sample fluid, while the spectrallabels can be used to identify the probe within the fluid (and hence theassociated moiety). As the individual probes can be quite small, and asthe number of spectral codes which can be independently identified canbe quite large, large numbers of individual assays might be performedwithin a single fluid sample by including a large number of differingprobes. These probes may take the form of quite small beads, with eachbead optionally including a spectral label, a moiety, and a bead body ormatrix, often in the form of a polymer. The reaction times and rareevent identification accuracy of such beads may be quite advantageous,particularly when the beads are free-floating within a fluid, withoutbeing affixed to a surface.

Together with their substantial advantages, there will be significantchallenges in implementing multiplexed, spectrally encoded bead-basedassay techniques. In particular, determining multiplexed assay resultsby accurately reading each spectral barcode and/or assay result fromwithin a fluid may prove quite problematic.

In light of the above, it would generally be desirable to provideimproved systems and methods for sensing and identifying signalgenerating bodies. It would be particularly beneficial if these improvedtechniques facilitated the identification of a plurality of spectralcodes generated by bodies disposed within and/or exposed to a fluid. Totake advantage of the potential capabilities of spectral coding ofmultiplexed assay probes, it would be highly desirable if these enhancedtechniques allowed detection and/or identification of large numbers ofspectral codes and/or other signals in a repeatably, highly timeefficient manner, while providing improved flexibility, ease of use,rare event/condition detection, and/or accuracy.

SUMMARY OF THE INVENTION

The present invention generally provides improved devices, systems,kits, and methods for detecting and/or identifying a plurality ofspectrally labeled bodies. The invention is particularly well-suited foridentifying particulate probes or “beads” which have been released in afluid so as to perform a multiplexed assay. These beads can be quitesmall and may have differing analytical properties, so that theiridentification may be problematic if standard inventory systems areapplied. By spectrally labeling the beads (for example, by includingmaterials which generate identifiable spectra associated with a bead ofa particular type in response to an excitation energy), a plurality ofbeads may be identified within the fluid. Such spectral labels may, forexample, comprise a plurality of differing markers (such assemiconductor nanocrystals, Raman scattering materials, or the like),with the differing markers each generating an identifiable signal, andthe combined signals for each type of probe defining the overallspectrum. These spectrally labeled beads have a wide range ofapplications, and are particularly beneficial for use in multiplexedassay systems.

The techniques of the present invention will often involve spatiallypositioning or restraining spectrally labeled bodies such as beads. Thebeads may be dynamically restrained by “sweeping” the fluid with anenergy beam such as a focused laser used as an optical tweezers.Alternatively, a plurality of beads may be spatially restrained withinan array of openings. Regardless, it will often be advantageous tooptically image the restrained bead using an optical train having aspectral dispersion element, such as a prism, a refractive grating, atransmissive grating, or the like.

In a first aspect, the invention provides a spectral labelidentification method comprising spatially restraining a firstspectrally labeled body. A first spectrum is generated from the firstbody while the first body is spatially restrained. The first spectrum isdispersed across a sensor surface, and the first body is identified fromthe first dispersed spectrum.

The spectrum generating step will often be performed by at least onesemiconductor nanocrystal, often by a plurality of semiconductornanocrystals, and preferably by a plurality of differing semiconductornanocrystal populations. Each semiconductor nanocrystal (or populationthereof) can act as a marker generating a signal having clearly definedsignal characteristics. The combination of these differing markers candefine the label, with the differing signals combining to define theoverall spectrum. When used as assay probes, spectrally encoded bodiesmay also include a moiety such as a selective affinity molecule, amember of a binding pair, or the like. The presence or absence of thespectrally encoded body may comprise an assay result signal, or anadditional assay signal may be generated by interaction of the moietywith an identifiable substance. Typically, a plurality of spectrallylabeled bodies are released in a fluid, and the first body is spatiallyseparated from the other released bodies when the first spectrum isgenerated. Ideally, a plurality of bodies are spatially restrained as anarray in which the bodies are separated from each other to facilitateidentification.

In many embodiments, a plurality of spectra from the bodies aresimultaneously dispersed across the sensor surface. An array of sitesmay be spaced to avoid excessive overlap of the dispersed spectra sothat each of the bodies can be identified from the associated spectrum.In some embodiments, the spectra may be sensed sequentially with ascanning system by moving a sensor field between the bodies. Suchsequential scanning may be used while simultaneously spatiallypositioning a plurality of bodies, and/or by sequentially restrainingthe bodies.

The bodies may be restrained within openings in a support structure,with the openings sized as to accommodate a single body. This caninhibit confusion by avoiding generating signals with two bodies whichare in (or near) contact. In some embodiments, the bodies may be drawninto the openings from the fluids by pumping fluid into the openings.This allows the bodies to be scanned in a fixed array configuration, andthen expelled to make room for a subsequent array. In alternativeembodiments, the labeled bodies may be sequentially spatially restrainedby an energy beam, such as by a focused laser beam using opticaltweezing techniques.

In many embodiments, assay signals may also be sensed from the bodies,the assay signals indicating results of an assay associated with thebodies. Multiplexed assays using these techniques may make use of about100 different bodies which can be independently identified from theirassociated spectra, with very highly multiplexed assays often includingat least 1,000 different bodies, optionally being 10,000 differentbodies.

In another aspect, the invention provides a method comprising spatiallyrestraining a plurality of spectrally labeled bodies so as to define anarray. A spectrally dispersed image of the array of bodies is directedonto a sensor to sense spectra generated by the bodies. The bodies areidentified from the spectra sensed by the sensor.

The bodies may be restrained within an array of openings affixed in amulti-well plate. The array of bodies may optionally be drawn into thearray of openings by drawing fluid into the openings. The bodies mayalso be expelled from the openings by expelling fluid from the openings.Another array of bodies may then be drawn into the openings by againdrawing fluid into the openings. In other embodiments, the bodies may berestrained in the array by an array of discreet binding sites. Thebinding sites may comprise a material capable of binding to the bodies.Suitable binding materials may comprise a nonspecific “sticky” oradhesive material, a member of a binding pair (with the other memberaffixed to the body), a selective affinity molecule which selectivelybinds to one or more particular bodies, or the like. Regardless ofwhether the array is defined by openings or binding sites, the arraywill preferably be arranged so as to inhibit the presence of more than asingle body at the sites of the array.

In another aspect, the invention provides a method comprising releasinga plurality of bodies in a fluid. A first body is spatially restrainedwithin the fluid by transmitting restraining energy through the fluidtoward the body. A first spectrum is generated from the spatiallyrestrained first body, and the first body is identified from the firstspectrum.

The spatially restraining step may be preformed with a focused laserbeam, particularly when the laser beam is used as an optical tweezers.The focused laser beam may be sized and configured to restrain a singlebody. For example, optical tweezers have a “trap” with a size determinedat least in part by the geometry of the focus laser beam. Where a sizeof the spectrally labeled body is at least about half the size of thetrap, the presence of a plurality of beads within the trap will beinhibited by spatial interference between the beads.

In some embodiments, the focused laser beam may be configured torestrain a plurality of the bodies simultaneously. For example, the trapmay be elongated so as to restrain the bodies along a line. This may beaccomplished by focusing the laser beam along a line segment.

In some embodiments, a separate excitation energy may be directed towardthe restrained body, with the body generating the spectrum in responseto the excitation energy. An alternative embodiments, the restrainedbody may generate a spectrum in response to the restraining energy.

The spectrum may be transmitted toward a sensor along an optical path.The restraining energy may be transmitted toward the body along at leasta portion of the optical path. Optionally, the restrained body may movewithin the fluid by moving the restraining energy or the fluid. Therestraining energy may sweep through the fluid to move the body toward afirst site, at which the spectrum may optionally be sensed. Therestraining energy may be swept through the fluid to move a second bodytoward a second site. The transmission of the restraining energy may beinhibited between the first and second sites so as to release the firstbody. Optionally, a series of bodies may be sequentially swept towardthe first site for sequential sensing of their spectra.

In another aspect, the invention provides a multiplexed assay systemcomprising a support structure having an array of sites. A plurality ofbodies each have a label for generating an identifiable spectrum inresponse to excitation energy. The bodies are restrainingly receivableat the sites. An optical train may image at least one site on a sensorsurface. The optical train includes a wavelength dispersive element.

In yet another aspect, the invention provides a multiplexed assay systemcomprising a plurality of bodies released in a fluid. The bodies havelabels for generating identifiable spectra. An energy transmitter iscoupled to the fluid so as to spatially restrain at least one body witha restraining energy beam. A sensor is oriented to reserve the spectrumfrom the at least one body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates an imaging system and high-Throughputassay method according the principles of the present invention.

FIG. 1A schematically illustrates an exemplary processor for the systemof claim 1.

FIG. 2 schematically illustrates probes having spectral labels and assaymarkers, in which the probes comprise bead structures disposed within atest fluid.

FIGS. 2A 2E schematically illustrate spectral codes or labels having aplurality of signals.

FIG. 3 schematically illustrates a system and method for determining aspectrum from a relatively large object by use of an aperture.

FIG. 4 schematically illustrates a method and structure for determininga spectrum from a small object, such as an assay probe havingsemiconductor nanocrystal markers, without using an aperture.

FIGS. 5A and 5B schematically illustrate a system and method fordetermining absolute spectra from a plurality of semiconductornanocrystals by limiting the viewing field with an aperture and byspectrally dispersing the apertured image.

FIG. 5C schematically illustrates a fluid flow assay scanning system andmethod.

FIGS. 6A 6C schematically illustrate a plate for positioningsemiconductor nanocrystal assay probes, together with a method for theuse of positioned probes in multiplexed assays.

FIG. 7 schematically illustrates a method for reading the spectrallabels and/or identifying assay results using the probe positioningplate of FIG. 6C.

FIG. 8 schematically illustrates an energy beam for spatiallyrestraining and optionally moving a spectrally labeled body, with theenergy being used as an optical tweezers.

FIG. 9 schematically illustrates a method for using an energy beam tosweep along a surface of a test fluid so as to position one or morespectrally labeled beads.

FIG. 10 schematically illustrates the use of separate energy beams forspatially restraining and generating an identifiable signal of aspectrally labeled body.

FIG. 11 schematically illustrates dynamically arraying spectrallylabeled beads.

FIG. 12 schematically illustrates the selective excitation and readingof a spectrally labeled body movably disposed within a test fluid.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention generally provides improved devices, systems,methods, compositions of matter, kits, and the like for sensing andinterpreting spectral information. The invention is particularlywell-suited to take advantage of new compositions of matter which cangenerate signals at specific wavelengths in response to excitationenergy. A particularly advantageous signal generation structure for useof the present invention is the semiconductor nanocrystal. Other usefulsignaling structures may also take advantage of the improvementsprovided by the present invention, including conventional fluorescentdies, radioactive and radiated elements and compounds, Raman scatteringmaterials, and the like.

The invention can allow efficient sensing and/or identification of alarge number of spectral codes, particularly when each code includesmultiple signals. The invention may also enhance the reliability andaccuracy with which such codes are read, and may thereby enable the useof large numbers of spectral codes within a relatively small region.Hence, the techniques of the present invention will find advantageousapplications within highly multiplexed assays, inventory control inwhich a large number of small and/or fluid elements are intermingled,and the like.

Spectra Labeling

Referring now to Ha 1, an inventory system 10 includes a library oflabeled elements 12 a, 12 b, . . . (collectively referred to as elements12) and an analyzer 14. Analyzer 14 generally includes a processor 16coupled to a detector 18. An energy source 20 transmits an excitationenergy 22 to a sensing field within a first labeled element 12 a oflibrary 8. In response to excitation energy 22, first labeled element 12a emits radiant energy 24 defining a spectral code. Spectral code ofradiant energy 24 is sensed by detector 18 and the spectral code isinterpreted by processor 16 so as to identify labeled element 12 a.

Library 8 may optionally comprise a wide variety of elements. In manyembodiments, labeled elements 12 may be separated. However, in theexemplary embodiment, the various labeled elements 12 a, 12 b, 12 c, . .. are intermingled within a test fluid 34. A real imaging is facilitatedby maintaining the labeled elements on or near a surface. As usedtherein, “areal imaging” means imaging of a two-dimensional area. Hence,fluid 34 may be contained in a thin, flat region between planarsurfaces.

Preferably, detector 18 simultaneously images at least some of thesignals generated by elements 12 from within a two-dimensional sensingfield. In some embodiments, at least some of the spectral signals fromwithin the sensing field are sequentially sensed using a scanningsystem. Regardless, maintaining each label as a spatially integral unitwill often facilitate identification of the label. This discrete spatialintegrity of each label is encompassed within the term “spatiallyresolved labels.” Preferably, the spatial integrity of the beads and thespace between beads will be sufficient to allow at least some of thebeads to be individually resolved over all other beads, preferablyallowing most of the beads to be individually resolved, and in manyembodiments, allowing substantially all of the beads to be individuallyresolved.

The spectral coding of the present invention is particularly well-suitedfor identification of small or fluid elements which may be difficult tolabel using known techniques. Elements 12 may generally comprise acomposition of matter, a biological structure, a fluid, a particle, anarticle of manufacture, a consumer product, a component for an assembly,or the like. All of these are encompassed within the term “identifiablesubstance.”

The labels included with labeled elements 12 may be adhered to, appliedto a surface of, and/or incorporated within the items of interest,optionally using techniques analogous to those of standard bar codingtechnologies. For example, spectral labeling compositions of matter(which emit the desired spectra) may be deposited on adhesive labels andapplied to articles of manufacture. Alternatively, an adhesive polymermaterial incorporating the label might be applied to a surface of asmall article, such as a jewel or a component of an electronic assembly.As the information in the spectral code does not depend upon the aerialsurface of the label, such labels can be quite small.

In other embodiments, the library will comprise fluids (such asbiological samples), powders, cells, and the like. While labeling ofsuch samples using standard bar coding techniques can be quiteproblematic, particularly when a large number of samples are to beaccurately identified, the spectral codes of the present invention canallow robust identification of a particular element from among ten ormore library elements, a hundred or more library elements, a thousand ormore library elements, and even ten thousand or more library elements.

The labels of the labeled elements 12 will often include compositions ofmatter which emit energy with a controllable wavelength/intensityspectrum. To facilitate identification of specific elements from amonglibrary 8, the labels of the elements may include combinations ofdiffering compositions of matter to emit differing portions of theoverall spectral code. In other embodiments, the signals may be definedby absorption (rather than emission) of energy, by Raman scattering, orthe like. As used herein, the term “markers” encompasses compositions ofmatter which produce the different signals making up the overallspectra. A plurality of markers can be combined to form a label, withthe signals from the markers together defining the spectra for thelabel.

The present invention generally utilizes one or more signals from one ormore markers. The markers may comprise semiconductor nanocrystals, withthe different markers often taking the form of different particle sizedistributions of semiconductor nanocrystals having different signalgeneration characteristics. One or more markers may be combined to forma spectral label which can generate an identifiable spectrum defining aspectral code, sometimes referred to as “spectral barcodes.” Thesespectral codes can be used to track the location of a particular item ofinterest or to identify a particular item of interest.

In many spectral codes, the different signals will have varying signalcharacteristics which are used as elements of the code. For example,semiconductor nanocrystals used in the spectral coding scheme can betuned to a desired wavelength to produce a characteristic spectralemission or signal by changing the composition and/or size of thesemiconductor nanocrystal. Additionally, the intensity of the signal ata particular characteristic wavelength can also be varied (optionallyby, at least in part, varying a number of semiconductor nanocrystalsemitting or absorbing at a particular wavelength), thus enabling the useof binary or higher order encoding schemes. The information encoded bythe semiconductor nanocrystals can be spectroscopically decoded from thecharacteristics of their signals, thus providing the location and/oridentity of the particular item or component of interest. As usedherein, wavelength and intensity are encompassed within the term “signalcharacteristics.”

While spectral codes will often be described herein with reference tothe signal characteristics of signals emitted with discrete, narrowpeaks, it should be understood that semiconductor nanocrystals and othermarker structures may generate signals having quite differentproperties. For example, signals may be generated by scattering,absorption, or the like, and alternative signal characteristics such aswavelength range width, slope, shift, or the like may be used in somespectral coding schemes.

Semiconductor Nanocrystals

Semiconductor nanocrystals are particularly well-suited for use asmarkers in a spectral code system because of their uniquecharacteristics. Semiconductor nanocrystals have radii that are smallerthan the bulk exciton Bohr radius and constitute a class of materialsintermediate between molecular and bulk forms of matter. Quantumconfinement of both the electron and hole in all three dimensions leadsto an increase in the effective band gap of the material with decreasingcrystallite size. Consequently, both the optical absorption and emissionof semiconductor nanocrystals shift to the blue (higher energies) withdecreasing size. Upon exposure to a primary light source, eachsemiconductor nanocrystal distribution is capable of emitting energy innarrow spectral linewidths, as narrow as 20 30 nm, and with a symmetric,nearly Gaussian line shape, thus providing an easy way to identify aparticular semiconductor nanocrystal. The linewidths are dependent onthe size heterogeneity, i.e., monodispersity, of the semiconductornanocrystals in each preparation. Single semiconductor nanocrystalcomplexes have been observed to have full width at half max (FWHM) asnarrow as 12 15 nm. In addition semiconductor nanocrystal distributionswith larger linewidths in the range of 40 60 nm can be readily made andhave the same physical characteristics as semiconductor nanocrystalswith narrower linewidths.

Exemplary materials for use as semiconductor nanocrystals in the presentinvention include, but are not limited to group II VI, III V, and groupIV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP,GaAs, GaSb, InP, InAs, InSb, AIS, AIP, AlSb, PbS, PbSe, Ge, Si, andternary and quaternary mixtures or alloys thereof. The semiconductornanocrystals are characterized by their nanometer size. By “nanometer”size, it is meant less than about 150 Angstroms (A), and preferably inthe range of 12 150 A.

The selection of the composition of the semiconductor nanocrystal, aswell as the size of the semiconductor nanocrystal, affects the signalcharacteristics of the semiconductor nanocrystal. Thus, a particularcomposition of a semiconductor nanocrystal as listed above will beselected based upon the spectral region being monitored. For example,semiconductor nanocrystals that emit energy in the visible rangeinclude, but are not limited to, CdS, CdSe, CdTe, and ZnTe.Semiconductor nanocrystals that emit energy in the near IR rangeinclude, but are not limited to, InP, InAs, InSb, PbS, and PbSe.Finally, semiconductor nanocrystals that emit energy in the blue tonear-ultraviolet include, but are not limited to, ZnS and GaN. For anyparticular composition selected for the semiconductor nanocrystals to beused in the inventive system, it is possible to tune the emission to adesired wavelength within a particular spectral range by controlling thesize of the particular composition of the semiconductor nanocrystal.

In addition to the ability to tune the signal characteristics bycontrolling the size of a particular semiconductor nanocrystal, theintensities of that particular emission observed at a specificwavelength are also capable of being varied, thus increasing thepotential information density provided by the semiconductor nanocrystalcoding system. In some embodiments, 2 15 different intensities may beachieved for a particular emission at a desired wavelength, however,more than fifteen different intensities may be achieved, depending uponthe particular application of the inventive identification units. Forthe purposes of the present invention, different intensities may beachieved by varying the concentrations of the particular sizesemiconductor nanocrystal attached to, embedded within or associatedwith an item or component of interest, by varying a Quantum yield of thenanocrystals, by varyingly quenching the signals from the semiconductornanocrystals, or the like. Nonetheless, the spectral coding schemes mayactually benefit from a simple binary structure, in which a givenwavelength is either present our absent, as described below.

In a particularly preferred embodiment, the surface of the semiconductornanocrystal is also modified to enhance the efficiency of the emissions,by adding an overcoating layer to the semiconductor nanocrystal. Theovercoating layer is particularly preferred because at the surface ofthe semiconductor nanocrystal, surface defects can result in traps forelectron or holes that degrade the electrical and optical properties ofthe semiconductor nanocrystal. An insulting layer (having a bandpasslayer typically with a bandgap energy greater than the core and centeredthereover) at the surface of the semiconductor nanocrystal provides anatomically abrupt jump in the chemical potential at the interface thateliminates energy states that can serve as traps for the electrons andholes. This results in higher efficiency in the luminescent process.

Suitable materials for the overcoating layer include semiconductorshaving a higher band gap energy than the semiconductor nanocrystal. Inaddition to having a band gap energy greater than the semiconductornanocrystals, suitable materials for the overcoating layer should havegood conduction and valence band offset with respect to thesemiconductor nanocrystal. Thus, the conduction band is desirably higherand the valence band is desirably lower than those of the semiconductornanocrystal. For semiconductor nanocrystals that emit energy in thevisible (e.g., CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, GaAs) or near IR (e.g.,InP, InAs, InSb, PbS, PbSe), a material that has a band gap energy inthe ultraviolet regions may be used. Exemplary materials include ZnS,GaN, and magnesium chalcogenides, (e.g., MgS, MgSe, and MgTe). Forsemiconductor nanocrystals that emit in the near IR, materials having aband gap energy in the visible, such as CdS, or CdSe, may also be used.While the overcoating will often have a higher bandgap than the emissionenergy, the energies can be, for example, both within the visible range.The overcoating layer may include as many as 8 monolayers of thesemiconductor material. The preparation of a coated semiconductornanocrystal may be found in U.S. patent application Ser. No. 08/969,302filed Nov. 13, 1997, entitled “Highly Luminescent Color-SelectiveMaterials”; Dabbousi et al., J. Phys. Chem B., Vol. 101, 1997, pp. 9463;and Kuno et al., J. Phys. Chem., Vol. 106, 1997, pp. 9869. Fabricationand combination of the differing populations of semiconductornanocrystals may be further understood with reference to U.S. patentapplication Ser. No. 09/397,432, previously incorporated herein byreference.

It is often advantageous to combine different markers of a label intoone or more labeled body. Such labeled bodies may help spatially resolvedifferent labels from intermingled items of interest, which can bebeneficial during identification. These label bodies may comprise acomposition of matter including a polymeric matrix and a plurality ofsemiconductor nanocrystals, which can be used to encode discrete anddifferent absorption and emission spectra. These spectra can be readusing a light source to cause the label bodies to absorb or emit light.By detecting the light absorbed and/or emitted, a unique spectral codemay be identified for the labels. In some embodiments, the labeledbodies may further include markers beyond the label bodies. Theselabeled bodies will often be referred to as “beads” herein, and beadswhich have assay capabilities may be called “probes.” The structure anduse of such probes, including their assay capabilities, are more fullydescribed in U.S. patent application Ser. No. 09/259,982, previouslyincorporated by reference.

Fabrication of Labeled Beads

Referring now to FIG. 2, first and second labeled elements 12 a, 12 bwithin test fluid 34 are formed as separate semiconductor nanocrystalprobes. Each probe includes a label 36 formed from one or morepopulations of substantially mono-disperse semiconductor nanocrystals37. The individual populations of semiconductor nanocrystals will oftenbe mono-disperse so as to provide a sufficient signal intensity at auniform wavelength for convenient sensing of the various signals withinthe code. The exemplary probes further include assay markers 38,together with a probe matrix or body material 39, which acts as abinding agent to keep the various markers together in a structural unit.Assay markers 38 generate signals indicating results of an assay, andare schematically illustrated as semiconductor nanocrystals having atleast one moiety with selective affinity for an associated testsubstance 35 which may be present within sample fluid 34. Preparation ofthe spectrally encoded probes will now be described, followed by a briefdescription of the use and structure of assay markers 38.

A process for encoding spectra codes into label body materials using afeedback system can be based on the absorbance and luminescence of thesemiconductor nanocrystals in a solution that can be used to dye thematerials. More specifically, this solution can be used for encoding ofa plurality of semiconductor nanocrystals into a material when thatmaterial is a polymeric bead.

A variety of different materials can be used to prepare thesecompositions. In particular, polymeric bead materials are an appropriateformat for efficient multiplexing and demultiplexing of finite-sizedmaterials. These label body beads can be prepared from a variety ofdifferent polymers, including but not limited to polystyrene,cross-linked polystyrene, polyacrylic, polysiloxanes, polymeric silica,latexes, dextran polymers, epoxies, and the like. The materials have avariety of different properties with regard to swelling and porosity,which are well understood in the art. Preferably, the beads are in thesize range of approximately 10 nm to 1 mm, more preferably in a sizerange of approximately 100 nm to 0.1 mm, often being in a range from 50nm to 1,000,000 nm, and can be manipulated using normal solutiontechniques when suspended in a solution.

Discrete emission spectra can be encoded into these materials by varyingthe amounts and ratios of different semiconductor nanocrystals, eitherthe size distribution of semiconductor nanocrystals, the composition ofthe semiconductor nanocrystals, or other property of the semiconductornanocrystals that yields a distinguishable emission spectrum, which areembedded into, attached to or otherwise associated with the material.The semiconductor nanocrystals of the invention can be associated withthe material by adsorption, absorption, covalent attachment, byco-polymerization or the like. The semiconductor nanocrystals haveabsorption and emission spectra that depend on their size andcomposition. These semiconductor nanocrystals can be prepared asdescribed in Murray et. al., (1993) J. Am. Chem. Soc. 115:8706 8715;Guzelian et. al., (1996) J. Phys. Chem. 100;7212 7219; or InternationalPublication No. WO 99/26299 (inventors Bawendi et al.). Thesemiconductor nanocrystals can be made further luminescent throughovercoating procedures as described in Danek et. al., (1966) Chem. Mat.8(1):173 180; Hines et. al., (1996) J. Phys. Chem. 100:468 471; Peng et.al., (1997) J. Am. Chem. Soc. 119:7019 7029; or Daboussi et. al., (1997)J. Phys. Chem.-B, 101:9463 9475.

The desired spectral emission properties may be obtained by mixingsemiconductor nanocrystals of different sizes and/or compositions in afixed amount and ratio to obtain the desired spectrum. The spectralemission of this staining solution can be determined prior to treatmentof the material therewith. Subsequent treatment of the material (throughcovalent attachment, co-polymerization, passive absorption, swelling andcontraction, or the like) with the staining solution results in amaterial having the designed spectral emission property. These spectramay be different under different excitation sources. Accordingly, it ispreferred that the light source used for the encoding procedure be assimilar as possible (preferably of the same wavelength and/or intensity)to the light source that will be used for the decoding. The light sourcemay be related in a quantitative manner, so that the emission spectrumof the final material may be deduced from the spectrum of the stainingsolution.

A number of semiconductor nanocrystal solutions can be prepared, eachhaving a distinct distribution of sizes and compositions, andconsequently a distinct emission spectrum, to achieve a desired emissionspectrum. These solutions may be mixed in fixed proportions to arrive ata spectrum having the predetermined ratios and intensities of emissionfrom the distinct semiconductor nanocrystals suspended in that solution.Upon exposure of this solution to a light source, the emission spectrumcan be measured by techniques that are well established in the art. Ifthe spectrum is not the desired spectrum, then more of a selectedsemiconductor nanocrystal solution can be added to achieve the desiredspectrum and the solution titrated to have the correct emissionspectrum. These solutions may be colloidal solutions of semiconductornanocrystals dispersed in a solvent, or they may be pre-polymericcolloidal solutions, which can be polymerized to form a matrix withsemiconductor nanocrystals contained within. While ratios of thequantities of constituent solutions and the final spectrum intensitiesneed not be the same, it will often be possible to derive the finalspectra from the quantities (and/or the quantities from the desiredspectra.)

The solution luminescence will often be adjusted to have the desiredintensities and ratios under the exact excitation source that will beused for the decoding. The spectrum may also be prepared to have anintensity and ratio among the various wavelengths that are known toproduce materials having the desired spectrum under a particularexcitation source. A multichannel auto-pipettor connected to a feedbackcircuit can be used to prepare a semiconductor nanocrystal solutionhaving the desired spectral characteristics, as described above. If theseveral channels of the titrator/pipettor are charged or loaded withseveral unique solutions of semiconductor nanocrystals, each having aunique excitation and emission spectrum, then these can be combinedstepwise through addition of the stock solutions. In between additions,the spectrum may be obtained by exposing the solution to a light sourcecapable of causing the semiconductor nanocrystals to emit, preferablythe same light source that will be used to decode the spectra of theencoded materials. The spectrum obtained from such intermediatemeasurements may be judged by a computer based on the desired spectrum.If the solution luminescence is lacking in one particular semiconductornanocrystal emission spectrum, stock solution containing thatsemiconductor nanocrystal may be added in sufficient amount to bring theemission spectrum to the desired level. This procedure can be carriedout for all different semiconductor nanocrystals simultaneously, or itmay be carried out sequentially.

Once the staining solution has been prepared, it can be used toincorporate a unique luminescence spectrum into the materials of thisinvention. If the method of incorporation of the semiconductornanocrystals into the materials is absorption or adsorption, then thesolvent that is used for the staining solution may be one that issuitable for swelling the materials. Such solvents are commonly from thegroup of solvents including dichloromethane, chloroform,dimethylformamide, tetrahydrofuran and the like. These can be mixed witha more polar solvent, for example methanol or ethanol, to control thedegree and rate of incorporation of the staining solution into thematerial. When the material is added to the staining solution, thematerial will swell, thereby causing the material to incorporate aplurality of semiconductor nanocrystals in the relative proportions thatare present in the staining solution. In some embodiments, thesemiconductor nanocrystals may be incorporated in a different butpredictable proportion. When a more polar solvent is added, afterremoval of the staining solution from the material, material shrinks, orunswells, thereby trapping the semiconductor nanocrystals in thematerial. Alternatively, semiconductor nanocrystals can be trapped byevaporation of the swelling solvent from the material. After rinsingwith a solvent in which the semiconductor nanocrystals are soluble, yetthat does not swell the material, the semiconductor nanocrystals aretrapped in the material, and may not be rinsed out through the use of anon-swelling, non-polar solvent. Such a non-swelling, non-polar solventis typically hexane or toluene. The materials can be separated and thenexposed to a variety of solvents without a change in the emissionspectrum under the light source. When the material used is a polymerbead, the material can be separated from the rinsing solvent bycentrifugation or evaporation or both, and can be redispersed intoaqueous solvents and buffers through the use of detergents in thesuspending buffer, as is well known in the art.

The above procedure can be carried out in sequential steps as well. Afirst staining solution can be used to stain the materials with onepopulation of semiconductor nanocrystals. A second population ofsemiconductor nanocrystals can be prepared in a second stainingsolution, and the material exposed to this second staining solution toassociate the semiconductor nanocrystals of the second population withthe material. These steps can be repeated until the desired spectralproperties are obtained from the material when excited by a lightsource, optionally using feedback from measurements of the interimspectra generated by the partially stained bead material to adjust theprocess.

The semiconductor nanocrystals can be attached to the material bycovalent attachment, and/or by entrapment in pores of the swelled beads.For instance, semiconductor nanocrystals are prepared by a number oftechniques that result in reactive groups on the surface of thesemiconductor nanocrystal. See, e.g., Bruchez et. al., (1998) Science281:2013 2016; and Ghan et. al., (1998) Science 281:2016 2018, Golvinet. al., (1992) J. Am. Chem. Soc. 114:5221 5230; Katari et. al. (1994)J, Phys. Chem. 98:4109 4117; Steigerwald et, al. (1987) J. Am. Chem.Soc. 110:3046. The reactive groups present on the surface of thesemiconductor nanocrystals can be coupled to reactive groups present onthe surface of the material. For instance, semiconductor nanocrystalswhich have carboxylate groups present on their surface can be coupled tobeads with amine groups using a carbo-diimide activation step, or avariety of other methods well known in the art of attaching moleculesand biological substances to bead surfaces. In this case, the relativeamounts of the different semiconductor nanocrystals can be used tocontrol the relative intensities, while the absolute intensities can becontrolled by adjusting the reaction time to control the number ofreacted sites in total. After the bead materials are stained with thesemiconductor nanocrystals, the materials are optionally rinsed to washaway unreacted semiconductor nanocrystals.

Referring once again to FIG. 2, labeled elements 12 a, 12 b (here in theform of semiconductor nanocrystal probes) may be useful in assays in awide variety of forms. Utility of the probes for assays benefitssignificantly from the use of moieties or affinity molecules 35′, asschematically illustrated in FIG. 2, which may optionally be supporteddirectly by label marker 37 of label 36, by the probe body matrix 39, orthe like. Moieties 35′ can have selective affinity for an associateddetectable substance 35, as schematically illustrated by correspondenceof symbol shapes in FIG. 2. The probes may, but need not necessarily,also include an integrated assay marker 38. In some embodiments, theassay marker will instead be coupled to the probes by coupling ofdetectable substance 35 to moiety 35′. In other words, the assay marker38′ may (at least initially) be coupled to the detectable substance 35,typically by binding of a dye molecule, incorporation of a radioactiveisotope, or the like. The assay markers may thus be coupled to the probeby the interaction between the moieties 35′ and the test or detectablesubstances 35. In other assays, the assay results may be determined bythe presence or absence of the probe or bead (for example, by washingaway probes having an unattached moiety) so that no dedicated assaymarker need be provided.

In alternative embodiments, the material used to make the codes does notneed to be semiconductor nanocrystals. For example, any fluorescentmaterial or combination of fluorescent materials that can be finelytuned throughout a spectral range and can be excited optically or byother means might be used. For organic dyes, this may be possible usinga number of different dyes that are each spectrally distinct.

This bead preparation method can be used generically to identifyidentifiable substances, including cells and other biological matter,objects, and the like. Pre-made mixtures of semiconductor nanocrystals,as described above, are attached to objects to render them subsequentlyidentifiable. Many identical or similar objects can be codedsimultaneously, for example, by attaching the same semiconductornanocrystal mixture to a batch of microspheres using a variety ofchemistries known in the art. Alternatively, codes may be attached toobjects individually, depending on the objects being coded. In thiscase, the codes do not have to be pre-mixed and may be mixed duringapplication of the code, for example using an inkjet printing system todeliver each species of semiconductor nanocrystals to the object. Theuse of semiconductor nanocrystal probes in chemical and/or biologicalassays is more fully described in U.S. patent application Ser. No.09/259,982 filed Mar. 1, 1999, the full disclosure of which isincorporated herein by reference.

The semiconductor nanocrystal probes of FIG. 2 may also be utilized todetect the occurrence of an event. This event, for example, may causethe source from which energy is transferred to assay marker 38 to belocated spatially proximal to the semiconductor nanocrystal probe.Hence, the excitation energy from energy source 20 may be transferredeither directly to assay markers 38, 38′, or indirectly via excitationof one or more energy sources adjacent the semiconductor nanocrystalprobes due to bonding of the test substances 35 to the moiety 35′. Forexample, a laser beam may be used to excite a proximal source such as asemiconductor nanocrystal probe 38′ attached to one of the testsubstances 35 (to which the affinity molecule selectively attaches), andthe energy emitted by this semiconductor nanocrystal 38′ may then excitean assay marker 38 affixed to the probe matrix.

Reading Beads

Referring once again to FIG. 1, energy source 20 generally directsexcitation energy 22 in such a form as to induce emission of thespectral code from labeled element 12 a. In one embodiment, energysource 20 comprises a source of light, the light preferably having awavelength shorter than that of the spectral code. Energy source 20 maycomprise a source of blue or ultraviolet light, optionally comprising abroad band ultraviolet light source such a deuterium lamp, optionallywith a filter. Energy source 20 may comprise an Xe or Hg UV lamp, or awhite light source such as a xenon lamp or a deuterium lamp, preferablywith a short pass or bandpass UV filter disposed along the excitationenergy path from the lamp to the labeled element 12 so as to limit theexcitation energy to the desired wavelengths. Still further alternativeexcitation energy sources include any of a number of continuous wave(cw) gas lasers, including (but not limited to) any of the argon ionlaser lines (457 nm, 488 nm, 514 nm, etc.), a HeCd laser, a solid-statediode laser (preferably having a blue or ultraviolet output such as aGaN based laser, a GaAs based laser with frequency doubling, a frequencydoubled or tripled output of a YAG or YLF based laser, or the like), anyof the pulsed lasers with an output in the blue or ultraviolet ranges,light emitting diodes, or the like.

The excitation energy 22 from energy source 20 will induce labeledelement 12 a to emit identifiable energy 24 having the spectral code,with the spectral code preferably comprising signals having relativelynarrow peaks so as to define a series of distinguishable peakwavelengths and associated intensities. The peaks will typically have ahalf width of about 100 nm or less, preferably of 70 nm or less, morepreferably 50 nm or less, and ideally 30 nm or less. In manyembodiments, a plurality of separate signals will be included in thespectral code as sensed by sensor 18. As semiconductor nanocrystals areparticularly well-suited for generating luminescent signals,identifiable energy 24 from label 12 a will often comprise light energy.To help interpret the spectral code from the identifiable energy 24, thelight energy may pass through one or more monochromator. ACharge-Coupled Device (CCD) camera or some other two-dimensionaldetector of sensor 18 can sense and/or record the images for lateranalysis. In other embodiments, a scanning system may be employed, inwhich the labeled element to be identified is scanned with respect to amicroscope objective, with the luminescence put through a singlemonochromator or a grating or prism to spectrally resolve the colors.The detector can be a diode array that records the colors that areemitted at a particular spatial position, a two-dimensional CCD, or thelike.

Information regarding these spectra from the labeled elements 12 willgenerally be transmitted as signals sent from sensor 18 to processor 16,the processor typically comprising a general purpose computer. Processor16 will typically include a central processing unit, ideally having aprocessing capability at least equivalent to a Pentium I® processor,although simpler systems might use processing capabilities equivalent toa Palm® handheld processor or more. Processor 16 will generally haveinput and output capabilities and associated peripheral components,including an output device such as a monitor, an input such as akeyboard, mouse, and/or the like, and will often have a networkingconnection such as an Ethernet, an Intranet, an Internet, and/or thelike. An exemplary processing block diagram is schematically illustratedin HG. 1A.

Processor 16 will often make use of a tangible media 30 having amachine-readable code embodying method steps according to one or moremethods of the present invention. A database 32, similarly embodied in amachine-readable code, will often include a listing of the elementsincluded in library 8, the spectral codes of the labels associated withthe elements, and a correlation between specific library elements andtheir associated codes. Processor 16 uses the information from database32 together with the spectrum characteristics sensed by sensor 18 toidentify a particular library element 12 a. The machine-readable code ofprogram instructions 30 and database 32 may take a wide variety offorms, including floppy disks, optical discs (such as CDs, DVDs,rewritable CDs, and the like), alternative magnetic recording media(such as tapes, hard drives, and the like), volatile and/or nonvolatilememories, software, hardware, firmware, or the like.

As illustrated in FIG. 1, methods for detecting and classifying spectrallabels (such as encoded materials and beads) may comprise exposing thelabels to light of an excitation source so that the semiconductornanocrystals of the label are sufficiently excited to emit light. Thisexcitation source is preferably of an energy capable of exciting thesemiconductor nanocrystals to emit light and may be of higher energy(and hence, shorter wavelength) than the shortest emission wavelength ofthe semiconductor nanocrystals in the label. Alternatively theexcitation source can emit light of longer wavelength if it is capableof exciting some of the semiconductor nanocrystals disposed in thematrix to emit light, such as using two-photon excitation. Thisexcitation source is preferably chosen to excite a sufficient number ofdifferent populations of semiconductor nanocrystals to allow uniqueidentification of the encoded materials. For example, using materialsstained in a 1:2 ratio of red to blue and a 1:3 ratio of red to blue, itmay not be sufficient to only excite the red emitting semiconductornanocrystals, (e.g., by using green or yellow light, of the sample inorder to resolve these beads). It would be desirable to use a lightsource with components that are capable of exciting the blue emittingand the red emitting semiconductor nanocrystals simultaneously, (e.g.,violet or ultraviolet). There may be one or more light sources used toexcite the populations of the different semiconductor nanocrystalssimultaneously or sequentially, but each light source may selectivelyexcite sub-populations of semiconductor nanocrystals that emit at lowerenergy than the light source (to a greater degree than higher energyemitting sub-populations), due to the absorbance spectra of thesemiconductor nanocrystals. Ideally, a single excitation energy sourcewill be sufficient to induce the labels to emit identifiable spectra.

Spectral Codes

Referring now to FIGS. 2A 2E, the use of a plurality of differentsignals within a single spectral label can be understood. In this simpleexample, a coding system is shown having two signals. A first signal hasa wavelength peak 40 a at a first discreet wavelength, while a separatesignal has a different wavelength peak 40 b. As shown in FIGS. 2A 2D,varying peak 40 b while the first peak 40 a remains at a fixed locationdefines a first family of spectral codes 1 a through 4 a. Moving thefirst peak 40 a to a new location allows a second family of spectralcodes to be produced, as can be understood with reference to FIG. 2E.

The simple code system illustrated in FIGS. 2A 2E includes only twosignals, but still allows a large number of identifiable spectra. Morecomplex spectral codes having larger numbers of peaks can significantlyincrease the number of codes. Additionally, the intensities of one ormore of the peaks may also be varied, thereby providing still higherorder codes having larger numbers of separately identifiable members.

Spectra Code Reading Systems

In general, fluorescent labeling is a powerful technique for trackingcomponents in biological systems. For instance, labeling a portion of acell with a fluorescent marker can allow one to monitor the movement ofthat component within the cell. Similarly, labeling an analyte in abioassay can allow one to determine its presence or absence, even atvanishingly small concentrations. The use of multiple fluorophores withdifferent emission wavelengths allows different components to bemonitored simultaneously. Applications such as spectral encoding cantake full advantage of multicolor fluorophores, potentially allowing thesimultaneous detection of millions of analytes.

When imaging samples labeled with multiple chromophores, it is desirableto resolve spectrally the fluorescence from each discrete region withinthe sample. As an example, an assay may be prepared in which polymerbeads have been labeled with two different chromophores and the resultsof the assay may be determined by the ratio of the two types of beadswithin the final sample. One could imagine immobilizing the beads andcounting each of the colors. Electronic imaging may involve a techniquefor acquiring an image of the sample in which spectral information isavailable at each discrete point. While the human eye is exceptionallygood at distinguishing colors, typical electronic photodetectors areoften effectively color-blind. As such, additional optical componentsare often used in order to acquire spectral information.

Many techniques might be combined with the present invention. Fouriertransform spectral imaging (Malik et al. (1996) J. Microsc. 182:133;Brenan et al. (1994) Appl. Opt 33:7520) and Hadamard transform spectralimaging (Treado et al. (1989) Anal. Chem 61:732A; Treado et al. (1990)Appl. Spectrosc. 44:1 4; Treado et al. (1990) Appl. Spectrosc. 44:1270;Hammaker et al. (1995) J. Mol. Struct. 348:135; Mei et al. (1996) J.Anal. Chem. 354:250; Flateley et al. (1993) Appl. Spectrosc. 47:1464);imaging through variable interference (Youvan (1994) Nature 369:79:Goldman et al. (1992) Biotechnology 10:1557); acousto-optical (Mortensenet al. (1996) IEEE Trans. Inst. Meas. 45:394; Turner et al (1996) Appl.Spectrosc. 50:277); liquid crystal filters (Morris et al. (1994) Appl.Spectrosc. 48:857); or simply scanning a slit or point across the samplesurface (Colarusso et al. (1998) Appl. Spectrosc. 52: 106A) are methodscapable of generating spectral and spatial information across atwo-dimensional region of a sample.

Referring now to FIG. 3, a system and method for reading spectralinformation from an arbitrarily large object 50 generally makes use of adetector 52 including a wavelength dispersive element 54 and a sensor56. Imaging optics 58 image object 50 onto a surface of sensor 56.Wavelength dispersive element 54 spectrally disperses the image acrossthe surface of the sensor, distributing the image based on thewavelengths of the image spectra.

As object 50 is relatively large when imaged upon sensor 56,differentiation of the discreet wavelengths within a spectrum 60 isfacilitated by the use of an aperture 62. As aperture 62 allows only asmall region of the image through wavelength dispersive element 54, thewavelength dispersive element separates the image components based onwavelength alone (rather than on a combination of wavelength andposition along the surface of image 50). Spectra 60 may then be directlydetermined based on the position of the diffracted image upon sensor 56,together with the intensity of image wavelength components as measuredby the sensor.

Referring now to FIG. 4, a spectra 60 of a spectrally labelednanocrystal bead 64 may be performed using a detector 66 without anaperture. As bead 64 has a signal generating area (as imaged by imagingoptics 58) which is much smaller than a sensing surface of sensor 56,bead 64 can act as a point-source of spectra 60. The various signals ofthe spectral code emanate from the small surface area of the bead, sothat the signal distribution across the sensor surface is dominated bythe wavelength dispersion, and no limiting of the image via an apertureis required. As used herein, a “true point source” is a light sourcewith a dimension which is at least as small as a minimum, diffractionlimited determinable dimension. A light source which is larger than atrue point source may “act” or be “used,” “treated,” or “analyzed” (orthe like) as a point source if it has a dimension or size which issufficiently small that its size acts like an aperture.

As described above, it will often be advantageous to include a pluralityof different spectrally labeled beads within a fluid. These labeledbeads will often be supported by the surrounding fluid, and/or will bemovable with the fluid, particularly in high-throughput multiplexedbead-based assays. Optionally, the beads may have a size sufficient todefine a suspension within the surrounding test fluid. In someembodiments, the beads may comprise a colloid within the test fluid. Insome embodiments, beads 64 may be movably supported by a surface of avessel containing the test fluid, for example, being disposed on thebottom surface of the vessel (where probe 64 has a density greater thanthat of the test fluid). In other embodiments, the beads may be affixedto a support structure and/or to each other. Still further alternativesare possible, such as for probe 64 to be floating on an upper surface ofthe test fluid, for the bead or beads to be affixed to or disposedbetween cooperating surfaces of the vessel to maintain the positioningof the bead or beads, for the bead or beads to be disposed at theinterface between two fluids, and the like.

As was described above, it will often be advantageous to includenumerous beads 64 within a single test fluid so as to perform aplurality of assays. Similarly, it will often be advantageous toidentify a large number of fluids or small discreet elements within asingle viewing area without separating out each spectral label from thecombined labeled elements. As illustrated in FIG. 4, the dispersedspectral image 68 of bead 64 upon sensor 56 will depend on both therelative spectra generated by the bead, and on the position of the bead.For example, bead 64′ is imaged onto a different portion 68′ of sensor56, which could lead to misinterpretation of the wavelengths of thespectra if the location of bead 64′ is not known. So long as anindividual bead 64 can be accurately aligned with the imaging optics 58and sensor system 66, absolute spectral information can be obtained.However, as can be understood with reference to FIG. 5A, a plurality ofbeads 64 will often be distributed throughout an area 70.

To ensure that only beads 64 which are aligned along an optical axis 72are imaged onto sensor 56, aperture 62 restricts a sensing field 74 ofthe sensing system. Where sensor 56 comprises an areal sensor such acharge couple device (COD), aperture 62 may comprise a slit aperture sothat spectral wavelengths .lamda. can be determined from the position ofthe dispersed images 68 along a dispersion axis of wavelength dispersiveelement 54 for multiple beads 64 distributed along the slit viewingfield 74 along a second axis y, as can be understood with reference toFIG. 5B. Absolute accuracy of the spectral readings will vary inverselywith a width of aperture slit 62, and the number of readings (and hencetotal reading time) for reading all the beads in area 70 will be longeras the slit gets narrower. Nonetheless, the beads 64 within thetwo-dimensional area 70 may eventually be read by the system of FIGS. 5Aand 5B with a scanning system which moves the slit relative to beads 64(using any of a variety of scanning mechanisms, such as movable mirrors,a movable aperture, a flow of the beads passed a fixed aperture, or thelike).

Sequential sensing of the spectra may be performed by moving theaperture relative to the sensing field, by software, by moving the beads(or other signal sources) relative to the optical train or scanningsystem, or even by scanning one of an excitation energy or the beadsrelative to the other. Aperture scanning may be effected by agalvanometer, by a liquid crystal display (LCD) selective transmissionarrangement, by other digital arrays, or by a digital micro-mirror array(DMD). Bead scanning systems may also use a fluid flow past a slitaperture, with the beads flowing with the fluid. Such bead flow systemsresult in movement of the aperture relative to the beads, even when theaperture remains fixed, as movement may be determined relative to thebead's frame of reference.

Referring now to FIG. 50, a simple fluid-flow assay system can make useof many of the structures and methods described herein above. In theillustrated embodiment, a test fluid 34 flows through a channel 131 sothat beads 64 move across sensing field 74. Beads 64 within theslit-apertured sensing region are spectrally dispersed and imaged asdescribed above. As the location of the slit-aperture is known, absolutespectral information regarding the label spectra and assay signals maybe determined from dispersed image 68. When a plurality of beads arewithin sensing region 74 but separated along the x axis as shown,multiple beads may be read simultaneously by a CCD, or the like. Flowingof the beads sequentially through sensing region 74 may allowsimultaneous assay preparation and reading using flow injection analysistechniques, or the like.

Imaging of sensing region 74 may be facilitated by providing a thin,flat channel 131 so that beads 64 are near opposed major surfaces of thechannel, with at least one of the channel surfaces being defined by amaterial which is transparent to the spectra and marker signals. Thisfluid-flow system may be combined with many aspects of the systemsdescribed hereinabove, for example, by providing two different energysources for the label spectra and assay markers, by areal imaging ofbeads 64 distributed throughout a two-dimensional sensing regionadjacent to or overlapping with slit-apertured sensing region 74, andthe like.

Restrained Position Beads

Techniques to analyze bead-based assays can be flow based and/or imagingbased. In the flow-based analysis, an instrument such as sheath flowcytometer is used to read the fluorescence and scatter information fromeach bead individually. Flow methods can have the disadvantage ofrequiring a relatively large volume of sample to fill dead volume in thelines and may complicate averaging or re-analysis of data points. Flowmethods allow a large number of beads to be analyzed from a givensample. Imaging based systems, such as the Biometric Image™ system, scana surface to find fluorescence signals. Advantages over the flow systemmay include small (<20 microliter) sample volumes and the ability toaverage data to improve signal to noise. A potential disadvantage is theuse of a large area in order to keep beads separated, and the dependenceon beads being at an appropriate dilution to ensure that a sufficientnumber can be analyzed without too many forming into doublets, triplets,or the like.

Referring now to FIGS. 6A 60, beads can be spatially restrained and/orimmobilized by a support structure 200 such as a planar surface.Typically, beads 64 will be restrained such that they are regularlyspaced in a chosen geometry or array 202. The beads can be immobilizedby restraining the beads in openings 204 in support structure 200, theopenings optionally comprising blind holes or wells. Such wells may befabricated by micromachining wells into the planar surface. For example,7-micron wells that are 7 microns deep, can be created by ablating a 7micron layer of parylene using a focused laser, and by affixing thelayer on a glass surface. Other methods can be used to createmicrostructures on the glass surface that behave as wells, includingelectron beam (or other particle) drilling, mechanical drilling, maskingand plasma etching, and the like.

The well dimensions may be chosen such that only a single bead iscaptured in the well and such that, when a lateral flow of fluid passesthe beads, the single beads remain trapped in the wells (see FIG. 60).For example, a 7-micron well may be suitable for analysis of beads fromaround 4 microns to 6 microns, or “monodisperse” 5 micron beads.

Other methods for capturing and spatially restraining beads includeselective deposition of polymers using light-activated polymerization,where the pattern of light is determined using a photoresist. Thepolymers then bind non-specifically to single beads and other beads canbe washed away. More generally, support surface 200 may optionallydefine array 202 as a discreet array of a material capable of affixingand/or bonding to beads 64. Suitable array site materials may comprise anon-specific “sticky” surface, such as those commercially available fromMOLECULAR MACHINES & INDUSTRIES, GMBH, of Heidelberg, Germany.Alternatively, the array material may comprise a specific bindingmoiety, a complimentary binding moiety of probes 64 typically defining abinding pair with the array material. For example, streptavidin maydiscreetly deposited on support structure 200 so as to define array 202,with biotinylated structures disposed on beads 64. Alternatively,streptavidin beads may specifically bind to biotinylated array sites ofthe support structure.

Regardless of whether array 202 is defined as a series of openings 204,or as a series of discreet bead binding sites, it will often beadvantageous to dispose the support structure 200 within a fluidcontainer, the support structure typically being affixed within at leastone well of a multi-well plate. In the exemplary embodiment, supportstructure 200 may comprise a glass structure bonded onto the bottom ofat least one well of a multi-well plate. A glass substrate 206 ofsupport structure 200 will preferably comprise a relatively thinstructure, typically being thinner than a thickness of a plate materialbordering the well containing the test fluid. By using multi-well plateshaving a clear material bordering the wells, and limiting the thicknessof a substrate 206, reading of beads 64 within openings 204 isfacilitated.

Layer 208 may be affixed to substrate 206, or may alternatively beaffixed directly to a surface of a multi-well plate, support structure200 typically being deposited along the bottom surface of the wellwithin a multi-well plate (or other test fluid containing volume) so asto allow gravity to help capture bead 64. Layer 208 may have a thicknessbetween about half and one and one half times a size of beads 64, withopenings 204 also often having a cross-sectional dimension between onehalf and one and one half times the size of the beads. Layer 208 maycomprise a material having signal transmission characteristics which aresignificantly different than that of the underlying substrate orcontainer material so as to enhance the accuracy or ease of reading thebeads. Still further structures might be used to immobilize and/orposition the beads, including superparamagnetic bead positioners beingdeveloped by IMMUNICON CORPORATION of Pennsylvania, and by ILLUMINA,INC. of San Diego, Calif.

In use, the mixture of spectrally encoded beads for a multiplexed assaycan be deposited onto the capture surface and allowed to settle intowells by gravity or to bind to the capture surface. Excess beads arethen washed away leaving single beads filling up some portion, forexample, >90% of the wells or capture positions.

The captured or spatially restrained beads can then be analyzed using animaging system as described above to capture fluorescence data atvarious emission wavelengths for each bead. This method providesadvantages over a simple scan of randomly placed beads because (1) beadsare known to be separated so the spatial resolution required fordetection can be reduced (as doublets do not have to be found andrejected). This leads to greater analysis efficiency, (2) the packing ofbeads can be considerably higher while still retaining spatiallyseparated singlet beads, (3) the beads do not move relative to thesupport and so can be scanned multiple times without concerns aboutmovement, and (4) the concentration of beads in the sample does not needto be precise (in the random scattering approach, too high aconcentration can lead to a high packing and eventually a multi-layerstructure, whereas too low a concentration leads to too few beads beinganalyzed).

In a system where spatial and spectral information are combined byplacing a coarse grating (reflection or transmission) in the emissionpath, such that the emitted light from each bead is spectrally dispersedin one dimension, the use of micromachined wells is particularly useful.The wells can be machined such that the dispersed images of each bead donot overlap. In addition, knowledge of the bead positions means thatabsolute wavelength determination can be carried out rather thanrelative determinations or using a spectral calibrator (See FIG. 7).

Referring now to FIGS. 6B and 7, array 202 may have a spacing 210, 212between array sites which is selected so as to avoid excessive overlapbetween spectra 214 as distributed across the imaging surface bydispersive element 54. More specifically, a spacing 210 aligned with adispersive axis 216 of dispersive element 54 will preferably besufficient so as to avoid any potential peak-to-peak overlap between thespectra. A spacing 212 transverse to dispersive element 216 may reduced,and/or the array pattern may be staggered so as to increase arraydensity while still avoiding overlap among the spectra. This facilitatescorrelation between the dispersed spectra and the known well/beadposition within array 202, as well as facilitating code interpretation.

Still further alternative bead positioning means are possible. In onevariation of the positioning wells illustrated in FIGS. 6A 6C, a closelypacked array of collimated holes may be distributed across a surface.Where the holes extend through a substrate defining the surface, apressure system may be provided along an opposed surface so as toactively pull beads 64 and test fluid 32 into the array of holes. Such asystem would allow a set of beads to be pulled into positioning wells,to have the assay results (optionally including bead labels and assaymarkers) read from the entrained beads, and then optionally, to push thebeads out of the through holes. Such a positioning and reading cycle maybe repeated many times to read a large number of beads within a testfluid. While there may be difficulty in transporting the beads and testfluid to the positioning surface, such a system has significantadvantages.

Spectrally encoded bodies or beads may be read by a variety of differingsystems. Optionally, a confocal excitation source may be scanned acrossa surface of a sample. Each time the excitation energy passes over anencoded bead, a fluorescence spectrum may be acquired. Byraster-scanning a point excitation source in both the X and Y dimensionsof a surface, all of the beads within a sample may be read sequentially.A disadvantage to such a system is that randomly arrayed beads mightrequire scanning substantially the entire sample with quite high spatialresolution to avoid missing a single bead. This may mean that asignificant amount of time is spent reading the sample surface or volumein regions that do not contain a bead. In addition, once a bead isfound, it should be scanned sufficiently to ensure that the spectrum isread from a similar portion of each bead, for example, from a center ofthe bead. If this is not done with sufficient accuracy, it may bedifficult to determine if a side of a bead, a center of the bead, or thelike has been read, inhibiting accurate assay label quantitation. Suchscanning is time consuming and significantly decreases the efficiency ofa point scanning system. As described above, an array of wells in, forexample, a slide glass may be used to organize the beads. By placing thebeads in an ordered array of wells, it is unnecessary to search for eachbead or to scan the beads carefully to find their centers. Rather, thearray of wells is simply registered to the reader, and the readercollects spectra from the center of each well. This can greatly increasethe rate of scanning, since no time is wasted searching for beads inplaces where they do not exist.

While the use of arrayed beads is appealing from a speed standpoint, itmay involve removing a sample fluid from a test container and placingthe sample in a specialized reading container structure having thewells. It would be highly desirable to be able to read the beadsdirectly in their original container, such as in the well of a 96-wellplate to avoid potential sample-to-sample cross contamination. Whilefabrication or modification of a standard 96-well plate is describedabove, it may be desirable to provide methods for restraining and/orremoving beads within a fluid. It may also be desirable to have atechnique that did not require one to look for beads in a region of thesample where no beads exist, while still allowing the beads to be readwithin a standard, unmodified 96-well plate or other standard sampleholder.

As can be understood with reference to FIGS. 8 12, the inventionprovides methods and systems for spatially restraining spectrallylabeled bodies. Optical tweezers can sweep spectrally encoded beads (orany other type of bead) into an ordered array, often as the beads areread. It is not necessary to order the beads prior to reading, as theymay optionally be organized as they are read.

Referring now to FIG. 8, optical tweezers 220 may comprise a laser beam222 focused by optics 224 to a light spot or focus region 226. Opticaltweezers often include a red or infrared laser beam 222, and may hold asmall body, such as a spectrally labeled probe or bead, at or near thecenter of the point of focus.

The force used to hold a body using optical tweezers may be lightpressure. Depending on the focal characteristics of optics 224, thesize, intensity, and other characteristics of laser 222, and the like, atrap 228 may be defined by optical tweezers 220, with the trap capableof spatially restraining beads 64 therein. The size of trap 228 may beselected so as to limit the size and/or number of beads 64 which can berestrained therein. For example, any bead smaller than approximately 10.mu.m in diameter that comes in contact with the focused spot 226 may bepulled into the point of highest intensity, as illustrated in FIG. 8. Ifthis point is moved in three-dimensional space, bead 64 may be moved aswell. Such movement of the bead within a moving optical tweezers isencompassed within the term “spatially restrained,” as used herein.However, for beads which are larger than about one half the size of trap228, or about 5 .mu.m in our example, only one bead can exist withintrap 228 at a time. Optical tweezers are a very standard, andsurprisingly simple tool, used in many different applications. See, e.g.Ashkin (1997) Proc. Natl. Acad. Sci. USA 94: 4853 4860; Helmerson et al.(1997) Olin. Chem: 43:379 383; Quake et al. (1977) Nature (London)388:151 154; Ashkin (1972) Sci, Amer. 226:63 71; and Ashkin (1970) Phys.Rev. Lett. 24:156 159. In the exemplary embodiments, optical tweezers220 will define a trap 228 having a dimension between about 0.1 and twotimes the size of bead 64, or between about 100 .mu.m and 100 .mu.m.

Referring now to FIGS. 9 and 11, optical tweezers 220 may be used tohold spectrally encoded beads in a moving or fixed position, and/or maybe used to order them in an array 202 for reading. Tweezers 220 may befocused near a surface 230 along which beads 64 are disposed, such asnear the bottom of a container 232. In the exemplary embodiment, thetweezers are focused near the bottom of a well of a multi-well plate.Beads 64 (optionally, but not necessarily being disposed within fluid34) are moved along surface 232 array sites 236, the beads ideally beingmoved to the center of a detection region of a point-scanning reader.Trap 228 may be scanned along surface 230 by moving optical tweezers220, by moving container 232 relative to the optical tweezers, bymanipulating optics 224 (for example, by moving a scanning mirror with agalvanometer, or the like), or the like.

Referring now to FIG, 10, excitation energy 22 and spatially restrainingenergy beam 222 may optionally comprise separate energies, such as alaser beam for spatially restraining beads 64 and a filtered white lightfor exciting semiconductor nanocrystals of beads 64 to generate anidentifiable spectrum. A dichroic mirror 240 may facilitate selectivelycombing energies having differing wavelengths, with the energy beamsoptionally being confocal, so as to define a substantially similar trapand excitation region. In the illustration of FIG. 10, the restrainingenergy beam and trap 228 are illustrated in solid lines, while theexcitation energy and excitation region are schematically illustratedwith dashed lines.

Referring now to FIG. 11, moving beads 64 relative to a surface 230 soas to prow an ordered array of beads 202 can be understood. To read allof the beads of an array 202, the scanner may efficiently be directedtoward a first array site 236 and then moved directly to a second arraysite, and so forth, skipping the empty space in between sites. Incontrast, if the disordered beads were to be point-scanned prior toordering the array of FIG. 11, only part of one of the three illustratedbeads 64 would be read, since this bead is not centered on the detectionregion or array site 236. This could lead to inaccurate assay results.Furthermore, after reading the first array site, the scanner might missthe remaining beads altogether, since they do not fall directly withinthe detection regions. Through use of optical tweezers 220 to order thearray of beads, reading of a plurality of beads is greatly improved.

If optical tweezers 220 are energized and oriented at a first array site236, an associated bead partially disposed within the array site may bepulled into the center of the trap, thereby providing an accuratequantitative measure of the assay label bead intensity by accuratelyalignment of a scanning system with the bead. After reading the firstbead, the tweezers may be turned off to release the first bead, and thescanner may advance to the right before the tweezers are again turnedon. If the scanner is moved sufficiently, retrapping of the first beadmay be inhibited prior to re-energizing the scanner. Once the tweezersare turned on again, the system may he moved to the intermediate spot ofthe simple array illustrated in FIG. 11. While no bead may be initiallypresent in this second array site 236, the process of scanning opticaltweezers 220 may pass an adjacent bead and this bead may be trapped andbrought into the intermediate array site. This second bead can then beread and released as before.

By using optical tweezers, time which might otherwise be spent lookingbetween the scanned spots may be used more efficiently. Rather, anybeads that fall in between spots may be pulled into a detection regionor array site. This technique can effectively integrate the area betweenscanned points by bringing any beads that are disposed between thepoints of an intermittent array scan into the next detection region.Since only one bead may be contained in the trap at a time, such asystem can significantly decrease the reading of multiple beadsco-located at an array site. Conveniently, if multiple beads lie betweenany two points, only one bead may be trapped by the optical tweezers andread. Choosing a scan distance that corresponds to the averageinter-bead spacing can decrease missing beads.

As can be understood with reference to FIG. 12, an alternativeembodiment may make use of optical tweezers which are separated from thesurfaces of a well. In this embodiment, the detection region remainslocated at the center of the trap. As the tweezers are turned on andoff, the solution is mixed, so that different beads may be brought intothe detection region and held while they are scanned. In other words,movement of fluid 34 relative to the reading system may optionally makeuse of a fixed optical tweezer and bead reading system. This techniquemay allow an identification or tracking system to scan a large number ofbeads without having to resort to precision scanning or control of theconcentration of beads along a bottom or other surface of a well. Hence,such a system may be for simpler and less expensive than a system havingscanning optics or the like. A similar system may restrain beads at areading site within a flow-based system, similar to that of FIG. 5C.

In other embodiments, the optical tweezers (and more specifically, thelaser beam) may be focused in a single dimension, for example, to a line(rather than being tightly focused in two dimension, for example, to apoint). Such a line-focused laser may create a trap region that extendsalong a line, rather than being spherically centered about a singlepoint. Such an optical tweezing system may be used to sweep or otherwisespatially restrain beads in distinct lines that can be scanned by a beadreader system having a slot aperture.

In many of the optical tweezer systems described above, the restrainingenergy beam may he an infrared laser, a red laser, or the like.Optionally, an infrared laser may be used which does not excite any ofthe semiconductor nanocrystals within a bead. In other embodiments, ared laser may be used that simultaneously traps the beads, and alsoexcites the marker semiconductor nanocrystals so as to generate theidentifiable spectrum. Optionally, the optical reading system may makeuse of at least a portion of the optical train of the optical tweezers.In other embodiments, the reading and restraining optical paths may beseparated.

The above-described spatial restraining optical tweezers, array ofopenings, and the like may be used to form microarrays within a volumeor surface of test structure, such as a multi-well plate for a varietyof assays. For example, any array-based assay could be processed anddetected within a multi-well plate without having to transfer the sampleonto an array surface. Detecting signals such as fluorescence from suchmicroarrays may be performed using any of the systems describedhereinabove. For example, all of the assays processed for a singlesemiconductor nanocrystal pathogen detection might be performed within awell of a multi-well plate, with the panel-array printed on the bottomof each well. Related techniques are described in U.S. patentapplication Ser. No. 60/182,844, entitled “Single Target Counting AssaysUsing Quantum Dot Nanoparticles” filed Feb. 6, 2000, the full disclosureof which is incorporated herein by reference. As part of the processing,appropriate analytes may be bound to a surface within a multi-wellplate, and the excess analyte removed prior to detection. This maydramatically simplify processing and detection. Other potentialapplications include sandwich immunoassays, DNA/RNA microarrays,surface-based molecular beacon arrays, and the like.

Specific structures for containing test fluids with beads, and/or fordirecting flows of such fluids and beads, may improve spectral codereading performance. Codes may be read from above, from below, or froman angle relative to vertical. Reading codes from below, for example,may be enhanced by using a fluid containing body with an opaque materialover the fluid. The fluid surrounding the beads may have an index ofrefraction which substantially matches that of the material of the lowerportion of the fluid containing body. Such structures may beparticularly beneficial when reading dense bead codes.

While the exemplary embodiments of the present inventions have beendescribed in some detail for clarity of understanding, a variety ofmodifications, adaptations, and changes will be obvious to those ofskill in the art. Hence, the scope of the present invention is limitedsolely by the appended claims.

1-16. (canceled)
 17. A method comprising: restraining a plurality ofspectrally labeled bodies with affinity binding so as to define amultiplexed array, wherein the spectrally labeled bodies are spatiallyresolved and comprise a plurality of fluorescent labels used to encodediscrete and different emission spectra wherein each emission spectrumhas a plurality of signals at differing wavelengths; directing an imageof the multiplexed array of bodies onto a sensor to sense spectragenerated by the bodies; and identifying the bodies from the spectrasensed by the sensor.
 18. The method of claim 17, wherein the bodies arerestrained to a support structure.
 19. The method of claim 17, whereinthe bodies are affixed to each other.
 20. The method of claim 18,wherein the support structure has a substantially planar surface. 21.The method of claim 18, wherein the support structure contains aplurality of openings sized to accommodate one of the spectrally labeledbodies.
 22. The method of claim 18, wherein a plurality of unboundbodies are washed away.
 23. The method of claim 17, wherein the bodiesare restrained within an array of openings affixed in a supportstructure.
 24. The method of claim 17, wherein the sensor is a CCDcamera.
 25. The method of claim 17, wherein the bodies are restrained inthe multiplexed array by an array of discrete binding sites.
 26. Amethod of claim 25, wherein the binding sites comprise a materialcapable of binding to the bodies.
 27. A method of claim 26, wherein thematerial comprises a selective affinity molecule which selectively bindsto one or more particular bodies.
 28. A method of claim 25, wherein thediscrete binding sites are arranged so as to inhibit a presence of morethan a single body at each binding site.
 29. A method of claim 17,wherein the spectrally labeled bodies include polymers.
 30. A method ofclaim 17, wherein the plurality of signals include a reference signal.31. A method of claim 30, wherein the reference signal identifies aspectral code family.
 32. A method of claim 17, wherein affinity bindingis facilitated by a chemical moiety.
 33. A method of claim 17, whereinthe affinity binding is specific.
 34. A method of claim 17, wherein theaffinity binding is facilitated with a binding pair.
 35. A method ofclaim 34, wherein one member of the binding pair is affixed to at leastone of the plurality of spectrally labeled bodies and the other memberis affixed to a site of the multiplexed array.
 36. The method of claim27, wherein the selective affinity molecule is streptavidin.
 37. Themethod of claim 27, wherein the selective affinity molecule is abiotinylated structure.